Abstract 2990: Antigen presentation deficiency and mesenchymal differentiation underlie resistance to immunotherapy in the murine syngeneic CT2A tumor model
Authors: Bryan Iorgulescu, Neil Ruthen, Ryuhjin Ahn, Eleni Panagioti, Prafulla Gokhale, Martha Neagu, Maria Speranza, Benjamin Eschle, Kara Soroko, Raziye Piranlioglu, Meenal Datta, Shanmugarajan Krishnan, Kathleen Yates, Gregory Baker, Rakesh Jain, Mario Suva, Donna Neuberg, Forest White, E. Chiocca, Gordon Freeman, Arlene Sharpe, Catherine Wu, David Reardon
Journal: Cancer Research (2023)

Abstract The GL261-luc2 and CT2A-luc syngeneic tumor lines are frequently used as immunocompetent orthotopic mouse models of human glioblastoma (huGBM), but demonstrate distinct differences in response to immune checkpoint blockade. Whereas GL261-luc2 is readily responsive to several immunotherapies, CT2A-luc is broadly resistant to diverse immunotherapeutic modalities. To decipher the cell-intrinsic mechanisms that drive immunotherapy resistance in CT2A-luc and to define the aspects of human cancer biology that these lines can best model, we systematically compared their genomic and phenotypic profiles. The transcriptional profiles of GL261-luc2 and CT2A-luc tumors resembled those of huGBM, despite neither line sharing the canonical genetic or histologic features of huGBM. Both models exhibited striking hypermutation and contained clonal hotspot mutations in RAS genes (Kras p.G12C in GL261-luc2 and Nras p.Q61L in CT2A-luc), which have only been identified in &amp;lt;1% of huGBM tumors. CT2A-luc distinctly displayed mesenchymal differentiation, upregulated angiogenesis, and multiple defects in antigen presentation machinery and interferon response pathways – confirmed at the genomic, transcriptomic, and proteomic levels. CT2A-luc uniquely contained multiple mutations in antigen presentation machinery genes that were computationally predicted to have deleterious biologic effects, including a clonal p.A275P missense mutation in Psmb8 (a subunit of the immunoproteasome, which degrades proteins into peptides for loading onto MHC class I) and a clonal p.Y488C missense mutation in Tap1 (which transports peptides into the endoplasmic reticulum for loading onto MHC class I). CT2A-luc also distinctly exhibited a single-copy loss of a chromosomal segment involving 4qC4 (FDR-adjusted p=0.04), which encompassed multiple type I IFN genes, as well as a single-copy loss of 10qD2-10qD3 (FDR-adjusted p=0.04), which contained Stat2, Stat6, and Ifng. Consistent with our observation of down-regulated IFN response pathways in CT2A-luc, phosphoproteomic analysis revealed decreased phosphorylation of several members of the JAK/STAT pathway in ex vivo CT2A-luc tumors, including Ptpn11 (i.e., Shp2), Il13ra1, and Stat3 - together suggesting reduced JAK/STAT signaling. Additionally, CT2A-luc demonstrated substantial baseline secretion of the CCL-2, CCL-5, and CCL-22 chemokines, all of which are known to play important roles as myeloid chemoattractants, in marked contrast to GL261-luc2. The defect in MHC class I expression could be overcome in CT2A-luc by interferon-γ treatment, which may underlie the modest efficacy of some immunotherapy combinations for CT2A-luc. Thus, CT2A-luc may be an informative preclinical model of immunotherapy resistance due to its mesenchymal differentiation and antigen presentation machinery deficits. Citation Format: Bryan Iorgulescu, Neil Ruthen, Ryuhjin Ahn, Eleni Panagioti, Prafulla Gokhale, Martha Neagu, Maria Speranza, Benjamin Eschle, Kara Soroko, Raziye Piranlioglu, Meenal Datta, Shanmugarajan Krishnan, Kathleen Yates, Gregory Baker, Rakesh Jain, Mario Suva, Donna Neuberg, Forest White, E. Chiocca, Gordon Freeman, Arlene Sharpe, Catherine Wu, David Reardon. Antigen presentation deficiency and mesenchymal differentiation underlie resistance to immunotherapy in the murine syngeneic CT2A tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2990.